Chem 265
Laboratory Exercises 5
The enzyme cellobiase
1. Background
Cellulose, the structural polysaccharide found in the cell walls of plants, is a source of sugar to organisms that produce
a family of enzymes known as cellulases. Cellulases catalyze the breakdown of cellulose to glucose. Humans and other
animals do not produce cellulases. Many plant eating animals are hosts to other organisms that do possess these
enzymes. For instance, termites have the protozoan Trichonympha living inside their gut. Trichonympha has a
bacterium called Rs-D17 living inside it that produces cellulase enzymes that break down cellulose, the main
component of wood. Ruminants, such as cows, harbor a team of anaerobic microorganisms that digest the plants they
eat. Bacteroidessuccinogenes is a common bacterium in the gut of cows that produces cellulases.
The biofuel industry uses cellulases to convert the cellulose in plant cell walls to sugars, such as glucose. The sugar
can then be converted to ethanol by microbial fermentation. This ethanol is also called cellulosic ethanol, which is
an efficient, more sustainable fuel to replace petroleum.
To understand the process of cellulosic ethanol production in detailwe need to study the structure of the plant cell
wall. Plant cell walls are made up of a variety of polysaccharides and other compounds, but the primary component is
cellulose. Cellulose molecules interact via hydrogen bonds to form cellulose microfibrils made up of 60–80 individual
strands of cellulose. Plant cell walls have additional molecules other than cellulose that contribute to their rigidity.
Hemicellulose and lignin are found in high quantities in the secondary cell walls of woody or fibrous plant tissue. For
cellulosic ethanol production, lignins must be removed because they inhibit enzymatic activity of cellulases.
Hemicelluloses must be cleaved from the cellulose to allow enzymatic breakdown of the cellulose.
The production of ethanol from plant material is a very complex procedure requiring multiple steps. Plant material is
first processed mechanically, as well as with acids or enzymes and heat to remove lignin. Once the lignin is removed,
the cellulose is more exposed and can be more readily broken down.
Cellulose is then broken down into glucose in three steps by three different types of enzymes.
• Endocellulases — These enzymes break down the internal bonds of the long chains of glucose molecules that form
cellulose.
• Exocellulases — These enzymes break the covalent linkages between the glucose units of cellulose that are on the
end of the cellulose molecules, releasing cellobiose.
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• Cellobiases (ß-glucosidases) — These enzymes break down the cellobiose left behind as a result of the work of the
first two enzymes.
2. The Enzyme Cellobiase
Cellobiasebreaks down cellobiose, a disaccharide made up of two glucose molecules connected together by a 1,4 β–
glucoside linkage. The breakdown of cellobiose by cellobiase is the final step in producing glucose from cellulose.
Although cellobiose is the natural substrate of cellobiase, there is no simple method to quantitatively detect the
product (glucose) or the disappearance of cellobiose. A simple colorimetric assay using an artificial substrate, pnitrophenylglucopyranoside, can be used to detect the enzymatic activity of cellobiase. The substrate pnitrophenylglucopyranoside is composed of a β-glucose covalently linked to a molecule of nitrophenol (see figure
below). When the bond connecting these two molecules is cleaved with the help of cellobiase, the p-nitrophenol is
released. To stop the activity of the enzyme and to create a colored product, the reaction mixture is added to a basic
solution. When the p-nitrophenol is placed in a basic solution, the hydroxyl group on the nitrophenolgets
deprotonatedand the nitrophenolate ion absorbs violet light (and reflects yellow light). This makes the solution yellow,
which can be quantitatedby using a spectrophotometer.
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An advantage of using a basic solution to develop the color of the p-nitrophenol is that the basic pH will also denature
the enzyme and stop the reaction.Using a spectrophotometer setup at a wavelength of 410 nm we will quantitate the
amount of nitrophenolate ion produced. JD will have prepared a standard curve with absorbance values of known
concentrations of p-nitrophenol.
3. Experiment 1: Measuring the rate of cellobiase activity and comparison to the rate of the uncatalyzed
reaction
You will compare the rate of breakdown of p-nitrophenylglucopyranoside to glucose and p-nitrophenol in in the
presence and absence of cellobiase. The enzyme and substrate are dissolved in a buffer that is at an ideal pH (pH 5.0)
for the reaction to occur. At set times, a sample of the enzyme reaction will be removed and added to a high pH stop
solution which will help develop the color of the product p-nitrophenol, as well as stop the reaction by increasing the
pH to above the range where the enzyme can work. By calculating how much p-nitrophenol is produced over time, the
rate of reaction can be calculated. By looking at small increments of time, you will be able to determine whether the
rate of the enzyme is constant or whether it slows down toward the end as the amount of substrate decreases.
Procedure
1. Locate the tubes labeled “Stop Solution”, “1.5 mM Substrate”, “Enzyme” and “Buffer”.
2. Label five cuvettes E1, E2, E4, E5, E6 (for five time points). Label only the upper part of the
cuvette’sside.
3. Label the two remaining cuvettes “Start” and “End” these cuvettes will serve as control time points at the start
and end of the reaction and neither cuvette will contain enzyme.
4. Pipet 500 µl of stop solution into each of the seven labeledcuvettes. The stop solution is a strong base, so avoid
getting it on your skin or clothes.
5. Label two empty conical tubes as “Enzyme Reaction” and “Control”.
6. Pipet2 ml of 1.5 mM substrate into the 15 ml conical tube labeled “Enzyme Reaction”. Pipet 2 ml of 1.5 mM
substrate into the conical tube labeled “Control”
7. Pipet 1000 µl of buffer into the 15 ml conical tube labeled “Control” and gently mix. Once you have mixed the buffer
with the substrate, remove 500 µl of this solution and add it to your cuvette labeled “Start”.
8. Pipet 1 ml of enzyme into the 15 ml conical tube labeled “Enzyme Reaction”. Gently mix as your partner STARTS THE
TIMER. This marks the beginning of the enzymatic reaction.
9. At the times indicated below, remove 500 µl of the solution from the “Enzyme Reaction” tube and add it to the
appropriately labeled cuvette containing the stop solution.
After 1min, remove 500 μl from the “Enzyme Reaction tube” and add them to cuvette E1. Mix the contents of the
cuvette.
After 2 min. do the same with E2; after 4 min with E4, after 5 min. with E5 and after 6 min. with E6. Mix the contents
of the cuvette thoroughly after each time point.
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10. After all the enzyme samples have been collected, remove 500 µl of the solution from the “C” reaction tube and
add it to the cuvette labeled “End”.
11. Use the spectrophotometer at a wavelength λ = 410 nm to measure the absorbance of the solutions in the 7
cuvettes. Use the sample S1 provided by JD to zero the spectrophotometer at 410 nm.Use the calibration curve
provided by JD (see below) to determine the corresponding p-nitrophenol concentrations.
Record your data in the following table (recall that 1 nanomol = 1 nm = 1 x 10-9mol)
Time
(minutes) cuvette
Enzyme
present
0
Start
NO
Absorbance calculated nm of
at
p-nitrophenol
410 nm
(in 1 ml of cuvette)
0.008
1
E1
YES
0.950
2
E2
YES
1.638
4
E4
YES
2.931
5
E5
YES
3.236
6
E6
YES
3.422
6
End
NO
0.003
Include your calculations for two of the time points in the space below.
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4. Experiment 2: Is there cellobiase in mushrooms?
1. JD will give you approximately 1 g a particular type of mushroom. Record the name and weight of the mushroom
sample.
Mushroom type: Baby Bella
Weight of the mushroom: ____0.010g
2. Place the mushroom in the mortar.
3. Add 2 ml of extraction buffer for every gram of mushroom into the mortar.
4. Using a pestle, grind the mushroom to a slurry
5. Transfer the slurry to an eppendorf tube and spin for 2 minutes at top speed. Make sure the centrifuge is balanced.
6. Transfer the supernatant to a clean eppendorf tube. This will be your mushroom extract. You will need at least 250
μl of mushroom extract to perform the enzymatic reaction.
7. Label 2 cuvettes as R (reaction) and C (control) and add 500 μl of stop solution to each cuvette.
8. Label 2 x 15 ml conical tubes as R and C.
9. Add 3 ml of 1.5 mM substrate to each tube.
10. Think for a minute what will you use as control and check your plan with JD. Record the components of your
control:
11. Set up your control tube C.
12. Setup your reaction tube R: pipet 250 μl of mushroom extract into the conical tube R and START YOUR TIMER!
After 5 min remove 500 μl from the reaction and add them to cuvette R.
13. Remove 500 μl from the control tube and add them to cuvette C.
14. Compare the absorbances of cuvettes C and R to decide whether you had cellobiase in your mushroom extract.
Absorbance of cuvette R: 0.288
Absorbance of cuvette C:0.014
Conclusion:
Analysis Questions
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1. Why is there no simple method to detect the formation of glucose or the disappearance of cellobiose?
2. Did you observe any changes in the enzyme reaction and control reaction tubes during the time that the reaction
was occurring?
3. What happened to the solution in each cuvette after you added the enzyme/substrate mixture to the stop solution?
4. Compare the amount of product produced in the enzyme-catalyzed reaction againstthe control where no enzyme
was added.
5. If you took a time point at 12 minutes, do you think more product would be produced than at 6 minutes? Explain.
6. Draw the plot of nanomoles of p-nitrophenol produced as a function of time. A hand-made sketch will not be
accepted. This has to be a graph prepared using Excel or at least a graph done using graph paper(like for a math
class) using a ruler, etc. Attach the graph to the lab report
7. Is the rate of product production constant over time? Hint: Is the slope of the line constant or does it change?
Justify your answer.
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8. Describe the experiments that you would need to do to determine KM and vmax for cellobiase. Full credit will not be
given to the answer “I will use a Lineweaver-Burke plot…”, aka, I need more details!
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